1. Field of the Invention
The present invention relates to a method of measurement in biological systems. More particularly, it relates to a method of quantifying molecular mixtures of and adhesions to minute amounts of biological substances using an accelerator mass spectrometer. Still more particularly, it relates to a process of measurement using intermediate and long lived isotopes bound to biological substances which are then converted to forms suitable for analysis by accelerator mass spectrometry.
Isotopes of various elements, particularly .sup.14 C, have been used in biological processes for some time as a means of tracing, to determine fate and speeds of reaction processes, and for other purposes.
The measurements are made by scintillation counters, autoradiography or other devices which measure the amount of decay of isotopes which have a relatively short half life.
These methods, in general, cannot be used where human beings are involved because of the potential radiation damage from the isotopes and the amount of sample required. At radiation levels which are not harmful to humans, decay counting methods are insufficiently specific and sensitive to give meaningful results. Moreover, the background contamination is high, creating problems for the users of the equipment.
2. The Prior Art
Isotopes are also used in biological systems for specific assays. The prior art of competition radio-immune assays (RIA) depends on the quantification of the number of radioactively labeled molecules which bind to their specific antibody in competition with an unknown amount of unlabeled molecules of the same chemical or class of chemicals. The number of labeled molecules binding to the antibodies is compared to the number of such molecules which bind to the antibody if no competitor molecules are present. This ratio of the bound, labeled molecules to the maximum possible number of bound molecules is then compared to a calibration curve to determine the number of unknown molecules which compete with the labeled molecules for binding locations on the antibody. This calibration curve is derived from similar measurements wherein known amounts of unlabeled molecules have been added to the solution of antibodies and labeled molecules.
These measurements depend on efficient separation of the antibodies and their bound molecules from the solution still containing labeled molecules. In the prior art, these antibodies are separated, for example, by immobilizing them on the walls or bottom of a tube or cell in which a known amount of the labeled chemical is added along with the unknown amount of chemical to be assayed. After the reactions of the molecules with the immobilized antibodies, the unbound reactants are separated from the antibodies by removal of the solution, followed by several rinses of the inside of the tube or well containing the immobilized antibodies. The tube or well is filled with or placed in a liquid scintillant and the radioisotope content is found using a scintillation counter. The sensitivity of a radio-immune assay is a function of the concentration of antibodies and radio-labeled molecules which are used. More sensitivity is gained by using lower concentrations of these reactants. These concentrations cannot be decreased substantially in the prior art of radio-immune assays, because the scintillation counters used to quantify the binding to the antibodies detect not only the bound radioisotopes but also spurious charged particles from cosmic rays or radioactive contaminations within the counter as well as the radio-labeled molecules which have bound directly to the surfaces of the tube or well without an intermediary antibody. These `background` events are indistinguishable from the particles which result from the decay of the radioisotopes in the molecules bound to the antibodies.
One way to increase sensitivity in an RIA is to use labelling isotopes which have short half-lives or decay times, such as isotopes of iodine. For a given amount of bound molecules, more counts will be detected above the counter background when shorter-lived isotopes are used. However, the concentration of these short-lived isotopes changes rapidly in time, so that chemical compounds incorporating these isotopes are used quickly after production. Calibration curves must be corrected often to account for this rapid decrease in the radioactivity of the labeled compounds. Further, these short-lived isotopes are often linked to the molecule in labile positions which allows them to deattach from the chemical compound of interest and cause another uncertainty in the quantification of the number of labeled molecules bound to antibodies.
A suggested solution of overcoming the problems associated with the use of short half life isotopes is to use an accelerator mass spectrometer.
As described by D. Elmore in an article in Biological Trace Element Research, Vol. 12, 1987, accelerator mass spectrometers can be used for a variety of purposes using long-lived radioisotopes. Such purposes include the introduction of isotopes as a tracer, then chemically processing the bulk tissue samples.
U.S. Pat. No. 4,037,100 describes an apparatus which can be used for the detection of electronegative particles and provide data as to their elemental composition. The apparatus includes an accelerator mass spectrometer (AMS) which can be used for making mass and elemental analyses. Still other references to AMS devices, and their uses include: Kilius et. al, "Separation of .sup.26 AL and .sup.26 Mg Isobars by Negative Ion Mass Spectrometry," Nature, Vol. 282, November 1979; A. E. Litherland, "Acceleration Mass Spectrometry," Nuclear Instruments and Methods in Physics Research B5, pp. 100-108, (1984); L. Brown, "Applications of Accelerator Mass Spectrometry," Ann. Rev. Earth Planet. Sci., Vol. 12, pp. 39-59, (1984); and A. E. Litherland, "Ultrasensitive Mass Spectrometry with Accelerators," Ann. Rev. Nucl. Part. Sci., Vol. 30, pp. 437-473, (1980).
Accelerator mass spectrometry (AMS) was developed as a highly sensitive method for counting long-lived but rare cosmogenic isotopes, typically those having half-lives between 10.sup.3 and 2.times.10.sup.7 years. Isotopes with this range of half-lives are too long-lived to detect easily by conventional decay counting techniques but are too short-lived on geological timescales to be present in appreciable concentrations in the biosphere or lithosphere. Assay of these cosmogenic isotopes (.sup.10 Be, .sup.14 C, .sup.26 Al, .sup.41 Ca, .sup.36 Cl, and .sup.129 I) by AMS has become a fundamental tool in archaeology, oceanography, and the geosciences, but has not been applied to problems of a biological or clinical nature.
It is an object of this invention to provide a method of biological analyses which is more specific than prior art methods.
It is a further object of this invention to provide a method of quantitive biological analysis which is more sensitive than methods known heretofore.
It is a still further object of this invention to provide a method of quantifying molecular mixtures of and adhesions to minute amounts of biological substances.
It is yet another object of this invention to provide a method of quantitive biological analysis using rare stable isotopes.
Another object of the invention is to provide a technique to measure the concentrations of long-lived radioisotopes at levels of a few parts in 10.sup.15 to parts in 10.sup.8 which can signal the presence or effects of very small amounts of labeled exogenous biochemicals within biological systems, organs, fluids, cells or parts of cells of living hosts, including humans.
Another object of the invention is to provide a technique to measure the concentrations of long-lived radioisotopes from within biological systems which does not make use of the radioactive decay of these isotopes.
Another object of the invention is to provide a technique to quantify the amount of an exogenous biochemical or several parts of an exogenous biochemical which have become adhered to or mixed with the natural biochemicals of a biological system using long-lived, radioactive molecular labels which are too low in concentration to be detected using techniques which depend on the decay of the radioisotopes.
Another object of the invention is to provide a technique to measure the concentrations of long-lived radioisotopes from within biological systems in which the labeled exogenous biochemical is stable over periods of time which are long compared to the period of biological effectiveness.
Another object of the invention is to provide a technique to measure the concentrations of long-lived radioisotopes from within biological systems in which the labeled exogenous biochemical is a close analogue of the natural, unlabeled form of the biochemical and without resort to the substitution of elements within the biochemical by short-lived radioisotopes of other similar elements or chemically labile short-lived radioisotopes.
Still another object of the invention is to provide a technique to measure the concentration of long-lived radioisotopes from within biological systems which represent molecular events whose probability is so low that natural levels of radioisotopes would mask the radioisotope labels attached to the exogenous effector.
These and other objects of the invention will be realized in the description, drawings, and claims to follow.